The object of this research project is to study the molecular biology of prothrombin synthesis and the role of vitamin K and the coumarin anticoagulant drugs in its regulation. The discovery of the new amino acid, gamma-carboxyglutamate in bovine prothrombin by Stenflo and associates (Proc. Natl. Acad. Sci. 71, 2730, 1974) has focussed studies of vitamin K action on phenomena related to carboxylation of a precursor peptide. The goals of this project are to 1) isolate, characterize and determine the amino acid sequence of residues 1-72 in the prothrombins and paraprothrombins from the rat and chick, 2) to determine the action of vitamin K in carboxylation of preprothrombin including a) requirements for the in vitro vitamin K-dependent formation of prothrombin in membrane-bound and soluble systems derived from liver microsomes and b) the role of "active forms" of vitamin K, 3) to translate the messenger RNA for preprothrombin in the heterologous system and identify the product in terms of size, susceptibility to degradation by various proteases, and amino acid sequence, 4) to isolate and characterize various components of the vitamin K-dependent carboxylase system from detergent solubilized membranes, and 6) to determine the basis of the interaction between warfarin and vitamin K in regulating preprothrombin carboxylation. The ultimate goal of this project is to reconstitute the vitamin K-dependent warfarin system for prothrombin synthesis from purified constituents and artificial phospholipid membranes.